- 1Gene Therapy Laboratory, School of Biosciences and Technology, VIT University, Vellore 632014, TN, INDIA
- 2Laboratory of Genetics, Salk Institute for Biological Studies, La Jolla, CA 92037, USA
- #Research Institute, SRM University, Chennai 603203, TN, INDIA (current affiliation)
Can J Biotech, Volume 1, Special Issue, Page 173, DOI: https://doi.org/10.24870/cjb.2017-a159
*Corresponding author: email@example.com
Patients with leukocyte adhesion deficiency type 1 (LAD1) suffer from recurrent bacterial infections due to mutations in the common β2 integrin subunit (CD18/ITGB2 gene). Treatment options include long-term administration of antibiotics, repeated granulocyte infusions, allogeneic bone marrow or stem cell transplantations, all of which have considerable limitations. Gene therapy could bring about a potential permanent cure for LAD1. We tested different fragment of the ubiquitous chromatin opening element (UCOE) from the human HNRPA2B1-CBX3 locus for their efficiency in driving the expression of human CD18 gene. Twelve new self-inactivating (SIN) lentiviral vectors were constructed, 10 of which incorporating various fragments of the UCOE, two others containing the long and short fragments of the elongation factor 1 alpha promoter (EF1αL, 1169 bp; EF1αS 248 bp) and a murine stem cell virus (MSCV) promoter within the context of the same lentiviral vector. These vectors were tested in vitro for the expression of human CD18 on the surface of CD34+ hematopoietic stem cells (HSCs) isolated from both moderate and severe LAD1 patients. Among the promoters tested in moderate patient’s CD34+ HSCs, 3’631 bp, 3’652 bp, 3’1262 bp, A2UCOE and EF1αS resulted in higher percentage of CD18+CD34+ cells (11.4% to 15.1% at MOI 10; 12.7% to 16.5% at MOI 100), comparable to the expression driven by the MSCV promoter (15.2% at MOI 10; 16.1% MOI 100). The 5’655 bp, 5’723 bp, 5’1296 bp, 2598 bp and EF1αL promoters resulted in comparatively lower levels of CD18 expression (10.4% to 11.1% at MOI 10; 5.7% to 10.9% at MOI 100). All the 3′ promoter fragments of the UCOE were further tested in a severe LAD1 patient’s CD34+ HSCs (1.6% to 5% at MOI 10 and 1% to 3.4% at MOI 100). Results obtained from this study would be useful in examining the human CD18 expression on murine LAD1 CD34+ HSCs in vitro followed by ex vivo studies to demonstrate the phenotypic correction of LAD1 in a murine model. Efforts are underway to compare the efficiency of gene correction using this conventional gene therapy approach with CRISPR-mediated gene correction of CD18 in human LAD1 CD34+ HSCs in vitro.